Alien sequences
a technology of alien sequences and sequences, applied in the field of alien sequences, can solve problems such as revealing defects in hybridization conditions that could affect the experimental outcom
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example 1
Identification of Alien Sequences
[0112] The present invention provides systems for identifying “alien” sequences that are not found in the relevant population of nucleic acids being hybridized to an array. For instance, the invention provides systems for identifying sequences that are not present in the cDNA of a selected organism.
[0113] In particular, a software program was developed that allows the user to generate “alien” cDNA's for a particular organism. The program, the algorithm of which was described above, takes in a list of all known cDNA sequences for that particular organism (e.g., mouse). From this list, the program calculates the codon frequency of the sequences as well as dinucleotide or transition sequences at the codon boundary. These files can be stored and are specific for the organism from which the frequencies are generated. The program then generates cDNA (with start and stop codons) using the above frequencies. A small percentage of the time (as may be specif...
example 2
Attaching Alien Sequences to Chips
[0116] Synthesis of alien oligonucleotides. Each of the 47 70mer alien oligonucleotide probes depicted in FIG. 2 was synthesized using an Expedite DNA synthetizer (Applied Biosystems, Framingham, Mass.) following standard protocols of phosphoramidite chemistry at a 200 mmol scale (S. L. Beaucage and R. P Iyer, Tetrahedron, 1992, 48: 2223-2311; S. L. Beaucage and R. P. Iyer, Tetrahedron, 1993, 49: 6123-6194). All alien oligonucleotides were modified at the 5′ terminus with a TFA-amino-C-6-phosphoramidite (Prime Organics, Lowell, Mass.) to enable subsequent covalent attachment of the oligonucleotide to a CodeLink (Amersham Biosciences) slide surface. After synthesis, oligonucleotides were cleaved and deprotected from the CPG support with concentrated ammonium hydroxide at 80° C. for 16 hours and lyophilized. The oligonucleotides were re-dissolved in 300 μL of water and then desalted on Performa SR DNA synthesis cleanup plates (EdgeBiosystems, Gaither...
example 3
Using Alien Gene Transcripts as In-Spike Controls
[0129] As described herein, one advantage of using alien sequences in microarray experiments is that their complements may serve as an in-spike control, enabling the experimenter to gauge the robustness of the target labeling and hybridization. Specifically, if an alien oligonucleotide is present on a chip or slide, then a known amount of its complement may be added to the population of nucleic acids (e.g., mRNA or cDNA) to be hybridized to the slide. The population, now spiked with a known amount of anti-alien nucleic acid, is then labeled and hybridized to the chip or slide. Global problems in labeling or hybridization are revealed through the extent of alien / anti-alien hybridization on the chip or slide.
[0130] In order to create an in-spike control that would mimic an experimental cDNA sample to the greatest extent possible, three alien genes have been designed to consist of four different 70mer alien sequences linked to one anot...
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